Study of the interference with A549 cell line by dual silencing neuroepithelial transforming gene-1 and vascular endothelial growth factor genes

Abstract

Objective To investigate the effects of both neuroepithelial transforming gene-1 (NET-1) and vascular endothelial growth factor (VEGF) genes silencing on the biological behavior and tube formation of human non-small cell lung cancer (NSCLC) cell line.Methods The designed and constructed single targeting small interfering RNA (siRNA) against the NET-1,VEGF,as well as dual genes targeting siRNA (DGT siRNA) against both NET-1 and VEGF genes were transfected into the lung adenocarcinoma cell line A549 respectively.After transfection,the mRNA and protein levels of NET-1 and VEGF were detected by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting.Methyl thiazol tetrazolium (MTT) assay was used to analyze the proliferation of A549,while Hoechst staining was carried out to detect cells apoptosis.The migrational and invasive capacity was estimated by transwell assay.Simultaneously,we extracted the supernatant of A549 cells after transfected for 48 h,and detected the VEGF concentration by enzyme linked immunosorbent assay (ELISA).Human umbilical vein endothelial cells (HUVECs) were incubated by the supernatant and the tube formation was detected.Results Compared with untreated cells (100.0％),the mRNA and protein expression levels of NET-1 were both downregulated in DGT siRNA group (53.3 ±5.6) ％ ; (42.9 ±5.0) ％ and NET-1 single siRNA group (35.8 ±5.1)％ ; (38.2 ± 1.6)％.Similarly,the mRNA and protein expression levels of VEGF were both downregulated in DGT siRNA group (47.9 ± 3.9) ％ ; (28.3 ± 2.2) ％ and VEGF single siRNA group [(49.4 ± 1.6)％ ; (18.5 ± 9.9) ％] compared with untreated group (100.0％) (P ＜ 0.05).Cell proliferation was both inhibited in single targeting siRNA and DGT siRNA by MTT assay,and was more obvious in DGT siRNA group (P ＜ 0.05).Numbers of cell permeating septum were significantly reduced in DGT siRNA group (25.63 ± 7.27) than those in NET-1 single siRNA group (35.70 ± 7.15) and VEGF single siRNA group (28.00 ±4.90) (P＜0.05).Higher rate of cell apoptosis was observed in DGT siRNA group (43.6 ± 5.0)％ than NET-1 single siRNA group (18.4 ± 4.8)％ and VEGF single siRNA group (9.6 ± 7.2) ％ by Hoechst staining (P ＜ 0.05).The VEGF concentration levels of A549 supernatant in DGT group (22.12 ±4.23) ng/L,NET-1 single siRNA group (108.00 ± 12.10) ng/L and VEGF single siRNA group (35.56 ± 7.40) ng/L were significantly decreased than untreated group (122.00 ± 10.31) ng/L.The tube numbers of HUVEC in DGT siRNA group (32.67 ± 4.50),NET-1 single siRNA group (48.33 ± 3.50) and VEGF single siRNA group (33.20 ± 2.65) were detected less than untreated group (73.43 ±4.58) (P ＜ 0.05),with DGT siRNA group showing much significant inhibitory effect than single siRNA groups.Conclusion Simultaneously silencing both NET-1 and VEGF genes can affect the biological behavior of A549 cells,promote cell apoptosis and reduce tube formation significantly.Multi-target genes intervention may become a new approach for prevention and treatment of lung cancer. Key words: RNA interference; Lung neoplasms; Neuroepithelial transforming gene-1 ; Vascular endothelial growth factor; Dual genes targeting